Pak. J. Bot., 48(3): 1183-1187, 2016.

CHARACTERIZATION OF FOUR SALSOLA SPECIES AND THEIR GENETIC RELATIONSHIP BY AFLP

AMAL M.E. ABDEL-HAMID1, 2

1Department of Biology, College of Sciences, Aljouf University, Sakaka, Saudi Arabia 2Department of Biological and Geological Sciences, Faculty of Education, Ain Shams University, Cairo, Egypt *Corresponding author’s email: [email protected]

Abstract

Amplified length polymorphism (AFLP) technique was used to characterize and detect molecular genetic markers for four Salsola species collected from Al Jouf region in the northern of Saudi Arabia and to shed light on their genetic relationships. Three primer combinations were used for AFLP analysis of the four Salsola species, they generated a total of 181 fragments of which 133 were species specific markers scored across Salsola species. The dendogram produced by Jaccard's coefficient and the UPGMA clustering method showed one main cluster, subdivided into two subclusters. The first sub cluster included Salsola schweinfurthii and Salsola tetrandra. The second sub cluster included Salsola villosa and Salsola cyclophylla. It is worth mentioning that this is the first study to use AFLP markers to characterize and detect molecular genetic markers for the four Salsola species and their genetic relationships.

Key words: Salsola, DNA fingerprinting, Genetic diversity, AFLP.

Introduction Flora of Saudi Arabia lacks the genetic and molecular studies; so, this study aimed to the definition Kingdom of Saudi Arabia has a varied heritage of of plant species, preservation of germplasm and to plant diversity as the climate is generally arid in most detect genetic relationships between different species parts of the country; nearly 30% of the flora is rare. of genus Salsola. Significant number of species and plant associations were found in the central and northern regions. Materials and Methods Flora of the northern regions, especially in Al Jouf region is dominated by annual species. However, Four species of the genus Salsola that considered as perennial components such as Salsola spp., Traganum important grazing plants in Saudi Arabia were chosen; nudatum, Haloxylon spp, Artemisia spp., Zilla spinosa, Salsola villosa Schult. , Salsola schweinfurthi Solms- etc. are forming large communities in all rage lands. The Laub.i, Salsola cyclophylla Baker and Salsola tetrandra climate is generally arid with an average annual rainfall of Forssk. They were collected from Al Jouf region in the 120 mm and an average maximum temperature of 48ºC northern of Saudi Arabia. The plant specimens have been during July to September and an average minimum identified following Migahid, 1996 and Al-Hassan, 2006. temperature of 8ºC during December-February. It is the first study to use AFLP technique to The genus Salsola belongs to the tribe Salsoleaes s. str. of the subfamily Salsoloideae S.1. (Kapralov et al., characterize and detect molecular genetic markers 2006; Akhani et al., 2007). A common name of various between the four species of Salsola and to shed light on members of this genus and related genera is saltwort, for their genetic relationship. their salt tolerance. The genus name Salsola is from the AFLP protocol: The original protocol of Vos et al., Latin salsus, meaning "salty"(Mosyakin, 2003). Salsoleae mainly distributed throughout arid and 1995 was used, while modifications suggested by semiarid regions in which these environments serve as Kamisugi et al., 2008; Huang & He, 2010; Song et al., favorable habitats for it due to the morphological, 2011 and Mikulaskova et al., 2012 were followed. anatomical, and physiological characters (Kadereit et al., Adaptors and primers sets were all synthesized by 2003).With the advance of molecular techniques, several Eurofins (Germany) (Table 1). types of DNA markers have been used like RFLP, RAPD, microsatellites (SSRs) and Amplified Fragment Length Table 1. Primers/adaptors names and sequence of primers Polymorphism (AFLP) to characterize species. at 5´→3´ used to establish the AFLP technique. AFLP technique is based on the complete Primer/Adaptor Sequence 5´ →3´ endonuclease restriction digestion of total genomic DNA, EcoRI– A1 CTCGTAGACTGCGTACC followed by selective PCR amplification resulting in EcoRI– A2 AATTGGTACGCAGTC unique, reproducible fingerprint for each individual and a Mse I– A1 GACGATGAGTCCTGAG large number of informative markers (Pompanin et al., Mse I– A2 TACTCAGGACTCAT 2005) and Arrigo et al., 2012). It is a very useful Eco + A GACTGCGTACCAATTCA technique that does not require prior information of DNA Mse + C GATGAGTCCTGAGTAAC sequence. It is able to detect high levels of polymorphism Eco + ACA FAM-GACTGCGTACCAATTCACA and resolve genetic relationships among groups that were Eco + AGG HEX-GACTGCGTACCAATTCAGG previously difficult to resolve with morphological Eco + ATA CY3-GACTGCGTACCAATTCATA characters or other molecular markers (Obae et al., 2013). Mse + CTC GATGAGTCCTGAGTAACTC

1184 AMAL M. E. ABDEL-HAMID

Preparation of adaptors and primers RL mix (0.2 µl) for every 25µl pre–selective PCR reaction was added. Micro–centrifuge tubes were placed a. Adaptors preparation: EcoR1 and Mse1 adaptors in a Flex Cycler (Analytic Jena) 96 Thermo-cycler. were re-suspended to 100µM with distilled water. After mixing the adaptors, they were heated at 95°C for 5 min Negative and positive controls were included. PCR to denature, and then left to cool slowly at room reactions were tested by 1% (1 g/100 ml) agarose gel temperature to re-nature completely for at least 1 hour. electrophoresis and visualized with 1X Red Safe® (5 µl/100ml), 5 µl of PCR reactions containing the 5 x b. Primers preparation: The primers were re-suspended GreenGoTaq® Flexi buffer were loaded onto the gel to 100 µM with distilled water. directly after amplification, additionally one lane was

Restriction R and ligation L of adaptors loaded with 2.5 µl of GeneRuler™ 100bp DNA ladder plus (Fermentas, SM1153). a. Preparation of RL mix: 10 µl of DNA were added to a Three different labeled PCR products of the same mix containing the restriction enzymes Msel (Biolabs, sample were mixed together (triplex analysis) as follow: R0525S), EcoR1 (Fermentas, ER0271), both Msel and EcoR1 adaptors, ATP (Fermentas, R0441), T4 ligase Selective PCR product To be added (Fermentas, EL0015) and Tango buffer (Fermentas, BY5) FAM labeled 2 µl that allow the activity of all the added enzymes. Finally HEX labeled 2 µl distilled water was added up to 20 µl. CY3 labeled 2 µl b. The RL reactions were incubated at 37°C for 4 hours Dist. water 4 µl and then placed on ice. Total 10 µl c. The RL reactions were tested by 1% (1g/100ml) agarose All samples were multiplexed and stored by order in gelelectrophoresis and visualized with 1X RedSafe®. Five ® a sterile 96–well PCR-plate and sent to the fragment µl of RL reactions containing the 5 xGreenGoTaq Flexi analysis service. buffer were loaded onto the gel, additionally one lane was loaded with 2.5 µlof GeneRuler™ 100bp DNA ladder plus AFLP data analysis: Band scoring and assortment by (Fermentas, SM1153). Peakscanner Applied biosystems & Microsoft Excel 2013 Pre-selective PCR: PCR reactions were performed using FAMD software (Schluter & Harris, 2006) was used to the adaptor specific primer with one base extension. A perform the phylogenetic analysis and Treegraph2 programs master mix was prepared and distributed depending on the to visualize the produced tree (Stover & Muller, 2010). number of PCR reactions. GreenGoTaq®Flexi DNA The analysis of the AFLP data was performed in two polymerase (Promega, M8295) was applied into 25µl known approachs. The first is based on the band binary mix/reaction. In sterile, nuclease–free micro-centrifuge criterion (i.e., codifying the detected bands to 1 when tubes, the following components were added on ice: presence and 0 when absence) and the other is based on the allele frequancy (i.e., number of a band presence relatively Stock Reaction Component to the number of all individuals). Each has its advantages concentration concentration and dis- advantages, therefore, both forms were combined GoTaq® Flexi buffer 5X 1X to obtain the maximum number of valuable indices MgCl2 25 mM 4 mM according to Bonin et al., 2007 methodology. dNTPs 10mM 0.2mM Eco + A primer 10µM 1µM Results Mse + C primer 10µM 1µM GoTaq™ 5 U/ µl 1.25 U Morphological characters: The four species are all RL mix - 2.0 µl perennial and small shrubs; however, there are some added up to 25 µl Distilled water - differences in the morphological characters between them total volume (Table 2).

Table 2. The differences of morphological characters among the four Salsola species. Salsola villosa Salsola schweinfurthii Salsola cyclophylla Salsola tetrandra Perennial Perennial Perennial Perennial Dwarf shrub Small shrub Dwarf shrub Dwarf shrub Stem height 70cm Stem height 20-50cm Stem height 20-40cm Stem height 60cm Old stem woody, young stems whitish Old stem woody, young stems Old stem woody, young stems fresh Woody purplish stem Stem and branches not striate purplish green Narrow, linear and alternate or Narrow, linear and alternate leaves sessile, Leaves with triple sites in nodal Opposite and succulent leaves, opposite, succulent leaves with terete or semiterete, bracts more or less branches, bracts more or less leaf- bracts more or less leaf-like and pointed tip, bracts more or less leaf- leaf-like and successively shorter like and successively shorter successively shorter like and successively shorter Glomerulate in bract axils, Paniculate spikes in forming a forming a spicate or paniculate Solitary, small and axillary flower Flowers in clusters and axillary spicate or paniculate inflorescence on inflorescence on upper part of upper part of branches bract axils branches flower Winged fruit Winged fruit Winged fruit Small winged fruit Collected from Hammad area Collected from Nayal valley Collected from Hammad area Collected from Nayal valley Heemstra et al. (1999), Collenette, (1999) and Al-Hassan (2006) CHARACTERIZATION OF SALSOLA SPECIES AND THEIR GENETIC RELATIONSHIP 1185

AFLP results: The AFLP analysis of the four Salsola number of species specific markers 22 was generated species with the three primer combinations generated a from primer combination Eco + ACA / Mse + CTC). total of 181 fragments (Table 3) of which 133 were (Table 3) polymorphic, corresponding to 71.90% level of polymorphism (Table 4). The number of total bands Genetic relationships: The genetic similarities among produced by each primer combination ranged from 35 to the four Salsola species based on Nei's method (Nei, 84 with an average of 60.33 bands. The percentage of 1978) and (Saitou & Nei, 1987). The highest pairwise polymorphism varied considerably among the primer similarity indices resulted from AFLP were between combinations. The highest ratio of polymorphism Salsola schweinfurthii and Salsola tetrandra, while the generated by Eco + ATA / Mse + CTC (79.03%), while lowest similarity indices were between Salsola the Eco + ACA / Mse + CTC produced the lowest schweinfurthii and Salsola cyclophylla. (Table 4). polymorphism (62.86%). The dendogram produced by Jaccard's coefficient and Species specific molecular markers for different the UPGMA clustering method showed one main cluster, Salsola species: The total number of species specific subdivided into two clusters (Fig. 1). Cluster 1 included markers scored across Salsola species is 133. Sixty two Salsola schweinfurthii and Salsola tetrandra. The second markers were positive species specific markers and 71 cluster combined Salsola villosa and Salsola cyclophylla. were negative species specific markers. The highest Distance scale bar is shown FAMD software (Schluter & number of species specific markers generated from AFLP Harris, 2006) was used to perform the phylogenetic analysis for a given combination was 62 (primer analysis and Treegraph2 programs to visualize the combination Eco + AGG / Mse + CTC), while the lowest produced tree (Fig. 1). (Stover & Muller, 2010).

Table 3. Number of total bands, polymorphic bands and polymorphism ratio of the four Salsola species generated by three AFLP primer combinations. Polymorphic bands Enzyme – primer combinations Total No. of bands Polymorphism % Species specific bands Non DNA markers Eco + ACA / Mse + CTC 22 13 35 62.86 Eco + AGG / Mse + CTC 62 22 84 73.81 Eco + ATA / Mse + CTC 49 13 62 79.03 Total 133 48 181 - Mean 44.33 24 60.33 71.90

Table 4. Similarity matrices based on molecular data for the four Salsola species generated by three AFLP primer combinations. Species Salsola villosa Salsola schweinfurthii Salsola cyclophylla Salsola tetrandra Salsola villosa 1.00 Salsola schweinfurthii 0.41 1.00 Salsola cyclophylla 0.46 0.39 1.00 Salsola tetrandra 0.45 0.56 0.53 1.00

Fig. 1. Neighbor-joining (NJ) tree based on Jaccard similarity coefficient between the four Salsola species. 1186 AMAL M. E. ABDEL-HAMID

The relationships among species were further Conclusions examined with principal coordinate analysis (PCoA). The PCoA analysis confirms the distribution of the grouping The results revealed that AFLP technique generated a produced by the UPGMA analysis. Four separate groups large number of polymorphisms so, it is considered to be were identified (Fig. 2). a useful tool for characterization and distinguishing between the different species of Salsola and giving a clear view about the genetic relationships between them.

Acknowledgments

I am grateful to Aljouf University, Sakaka, Saudi Arabia for funding this research under the project number 334/35 and appreciate to Prof. Dr. Eman Fahmy Department of Genetics, Faculty of Agriculture, Ain Shams University for revising the manuscript.

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(Received for publication 27 March 2015)